Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT

Kwok-Min Hui, George L Orriss, Tilman Schirmer, Bergljót Magnadóttir, Jürg A Schifferli, Jameel M Inal

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10 Citations (Scopus)


CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.

Original languageEnglish
Pages (from-to)863-8
Number of pages6
JournalBiochemical Journal
Issue numberPt 3
Publication statusPublished - 1 Aug 2005


  • Amino Acid Motifs
  • Binding Sites
  • Carrier Proteins
  • Complement C2
  • Complement C4
  • Complement Inactivator Proteins
  • Humans
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins
  • von Willebrand Factor
  • Journal Article
  • Research Support, Non-U.S. Gov't


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